Method for manufacturing composition for lowering blood lipid and elevating high-density lipoprotein

ABSTRACT

The present invention discloses a composition for lowering blood lipid and elevating high-density lipoprotein and a method for manufacturing the same; the composition comprises monascin or ankaflavin, or a combination thereof; the manufacturing method comprises the steps of: treating a  Monascus  fermented product with acetone for three times; elevating the concentration of the  Monascus  fermented product by a process of decompress concentration; and extracting the monascin and the ankaflavin from the  Monascus  fermented product with a silica gel column chromatography, a Sephadex LH-20 column chromatography, the silica gel column chromatography, and a preparative high performance liquid chromatography sequentially.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to composition for lowering blood lipidand elevating high-density lipoprotein, and more particularly to acomposition containing monascin or ankaflavin, or a combination thereof,for lowering blood lipid and elevating high-density lipoprotein.

2. Description of the Prior Art

The content of cholesterol in blood is closely related to the incidenceof cardiovascular diseases. According to previous studies, dyslipidemiaplays an important role in the cardiovascular diseases, thus theconcentration of cholesterol in blood can be a well predictor for thecardiovascular diseases. When the concentration of serum cholesterol ismore than 200 mg/dL, the probability of dying of coronary heart diseaseis raised rapidly. A person who has high content of cholesterol shouldhave high morbidity rate of coronary heart disease and high death rate,but if the content of cholesterol is lowered by the treatment of amedical or a non-medical process, the incidence rate of thecardiovascular diseases caused by coronary sclerosis can be decreasedsignificantly.

In recent years, health foods are developed vigorously, andmulti-functional Monascus fermented products are gradually paidattention. In Asia, the application of Monascus species in food andmedicine has thousand years of history.

The important secondary metabolites of Monascus species include: (1) agroup of pigments, including red pigments (rubropunctamine andmonascorubramine), yellow pigments (ankaflavin and monascin) and orangepigments (rubropunctanin and monascorubrin); (2) cholesterol-loweringsubstance, such as monacolin K (also called as lovastatin, mevinolin andmevacor); (3) blood pressure lowering substance, such as γ-aminobutyricacid (GABA); and (4) antioxidants, including dimerumic acid and3-hydroxy-4-methoxy-benzoic acid.

In the above important secondary metabolites of Monascus species, owingto the monacolin K can reduce the activity of hydroxylmethylglutaryl-CoAreductase (HMG-CoA reductase), which is a key enzyme in the process ofcholesterol biosynthesis, the monacolin K has a significantly effect forlowering cholesterol.

On the other hand, cholesterol in blood can be divided into twocategories according to their density, including High DensityLipoprotein-Cholesterol (HDL-C) and Low Density Lipoprotein-Cholesterol(LDL-C). Many researches indicate that HDL-C is a kind of goodcholesterol, and LDL-C is bad cholesterol. High concentration of HDL-Cand low concentration of LDL-C are helpful in decreasing the incidenceof cardiovascular disease and atherosclerosis.

Owing to total cholesterol (TC) is composed of HDL-C, LDL-C,triglyceride (TG), and other lipoprotein cholesterols, the amount ofHDL-C and LDL-C can be substantially increased in company with theelevation of the concentration of TC. Most researches illustrate thatafter the concentration of TC being lowered by cholesterol-loweringdrugs, the concentration of HDL-C is also decreased greatly. However, ifthe concentration of HDL-C can be maintained or elevated, the preventionof cardiovascular diseases can be achieved.

However, although the above-mentioned monacolin K has the effect oflowering cholesterol, the concentration of HDL-C can not be elevated bymonacolin K, and the effect of preventing cardiovascular diseases andatherosclerosis by monacolin K is very limited.

In view of this, it is necessary to provide a novel composition and amethod for manufacturing the same, wherein the composition has effectsof lowering blood lipid and elevating the concentration of HDL-Csimultaneously, so as to decrease the incidence of cardiovasculardiseases and atherosclerosis effectively.

SUMMARY OF THE INVENTION

In view of the above shortcomings of the prior art, the inventors of thepresent invention resorted to past experience, imagination, andcreativity, performed experiments and researches repeatedly, andeventually devised the present invention, a composition for loweringblood lipid and elevating high-density lipoprotein and a method formanufacturing the same.

The major objective of the present invention is to provide thecomposition for lowering blood lipid and elevating high-densitylipoprotein, which comprises monascin and can lower the concentration ofblood lipid and elevate the concentration of high-density lipoproteineffectively.

According to the above objective, the present invention provides acomposition for lowering blood lipid and elevating high-densitylipoprotein, wherein the composition comprises monascin, which is aMonascus yellow pigment and extracted from a Monascus fermented product.

Another objective of the present invention is to provide the method formanufacturing a composition for lowering blood lipid and elevatinghigh-density lipoprotein, by a series of extraction processes, acomposition containing monascin can be obtained, and the composition canlower the concentration of blood lipid and elevate the concentration ofhigh-density lipoprotein effectively.

According to the above objective, the present invention provides amethod for manufacturing a composition for lowering blood lipid andelevating high-density lipoprotein, wherein the method comprises thesteps of: (1) providing a Monascus fermented product; (2) treating theMonascus fermented product with acetone for three times; (3) elevatingthe concentration of the product obtained from the previous step by aprocess of decompress concentration in a specific temperature range; (4)separating a pigment fraction from the product obtained from the prevwusstep by a silica gel column chromatography; (5) separating a yellowpigment fraction from the pigment fraction by a Sephadex LH-20 columnchromatography; (6) separating a fraction containing monascin andankaflavin from the yellow pigment fraction by the silica gel columnchromatography; and (7) separating monascin from the fraction containingmonascin and ankaflavin obtained from the previous step by a preparativehigh performance liquid chromatography (pre-HPLC). “Decompressconcentration,” as used in the present disclosure, refers to a processto increase the concentration of a solute in a solution by reducing theair pressure over the solution below the ambient air pressure. Thereduced air pressure may be determined empirically.

Further objective of the present invention is to provide the compositionfor lowering blood lipid and elevating high-density lipoprotein, whichcomprises ankaflavin and can lower the concentration of blood lipid andelevate the concentration of high-density lipoprotein effectively.

According to the above objective, the present invention provides acomposition for lowering blood lipid and elevating high-densitylipoprotein, wherein the composition comprises ankaflavin, which is aMonascus yellow pigment and extracted from a Monascus fermented product.

Further objective of the present invention is to provide the method formanufacturing a composition for lowering blood lipid and elevatinghigh-density lipoprotein, by a series of extraction processes, acomposition containing ankaflavin can be obtained, and the compositioncan lower the concentration of blood lipid and elevate the concentrationof high-density lipoprotein effectively.

According to the above objective, the present invention provides amethod for manufacturing a composition for lowering blood lipid andelevating high-density lipoprotein, wherein the method comprises thesteps of: (1) providing a Monascus fermented product; (2) treating theMonascus fermented product with acetone for three times; (3) elevatingthe concentration of the product obtained from the previous step by aprocess of decompress concentration in a specific temperature range; (4)separating a pigment fraction from the product obtained from theprevious step by a silica gel column chromatography; (5) separating ayellow pigment fraction from the pigment fraction by a Sephadex LH-20column chromatography; (6) separating a fraction containing monascin andankaflavin from the yellow pigment fraction by the silica gel columnchromatography; and (7) separating ankaflavin from the fractioncontaining monascin and ankaflavin obtained from the previous step by apre-HPLC.

Further objective of the present invention is to provide the compositionfor lowering blood lipid and elevating high-density lipoprotein, whichcomprises monascin and ankaflavin and can lower the concentration ofblood lipid and elevate the concentration of high-density lipoproteineffectively.

According to the above objective, the present invention provides acomposition for lowering blood lipid and elevating high-densitylipoprotein, wherein the composition comprises monascin and ankaflavin,which are both Monascus yellow pigments and extracted from a Monascusfermented product.

Further objective of the present invention is to provide the method formanufacturing a composition for lowering blood lipid and elevatinghigh-density lipoprotein, by a series of extraction processes, acomposition containing monascin and ankaflavin can be obtained, and thecomposition can lower the concentration of blood lipid and elevate theconcentration of high-density lipoprotein effectively.

According to the above objective, the present invention provides amethod for manufacturing a composition for lowering blood lipid andelevating high-density lipoprotein, wherein the method comprises thesteps of: (1) providing a Monascus fermented product; (2) treating theMonascus fermented product with acetone for three times; (3) elevatingthe concentration of the product obtained from the previous step by aprocess of decompress concentration in a specific temperature range; (4)separating a pigment fraction from the product obtained from theprevious step by a silica gel column chromatography; (5) separating ayellow pigment fraction from the pigment fraction by a Sephadex LH-20column chromatography; (6) separating a fraction containing monascin andankaflavin from the yellow pigment fraction by the silica gel columnchromatography; (7) separating monascin and ankaflavin respectively fromthe fraction containing monascin and ankaflavin obtained from theprevious step by a pre-HPLC; and (8) mixing the monascin and theankaflavin according to a specific ratio.

BRIEF DESCRIPTION OF THE DRAWINGS

For a complete understanding of the aspects, structures and techniquesof the invention, reference should be made to the following detaileddescription and accompanying drawings wherein:

FIG. 1 is a flow chart diagram of a method for manufacturing a Monascusfermented product of the present invention;

FIG. 2 is a flow chart diagram of a method for manufacturing acomposition for lowering blood lipid and elevating high-densitylipoprotein according to a first preferred embodiment of the presentinvention;

FIG. 3 is a flow chart diagram of detailed steps of step 104 accordingto the first preferred embodiment of the present invention;

FIG. 4 is a flow chart diagram of detailed steps of step 105 accordingto the first preferred embodiment of the present invention;

FIG. 5 is a flow chart diagram of detailed steps of step 106 accordingto the first preferred embodiment of the present invention;

FIG. 6 is a flow chart diagram of detailed steps of step 107 accordingto the first preferred embodiment of the present invention;

FIG. 7 is a flow chart diagram of a method for manufacturing acomposition for lowering blood lipid and elevating high-densitylipoprotein according to a second preferred embodiment of the presentinvention;

FIG. 8 is a flow chart diagram of detailed steps of step 207 accordingto the second preferred embodiment of the present invention;

FIG. 9 is a flow chart diagram of a method for manufacturing acomposition for lowering blood lipid and elevating high-densitylipoprotein according to a third preferred embodiment of the presentinvention;

FIG. 10 is a flow chart diagram of detailed steps of step 307 accordingto the third preferred embodiment of the present invention;

FIG. 11 is a diagram of quantitative results of a lipid plaque stainingexperiment; and

FIG. 12 is a comparative diagram for the effect of the inhibition ofcholesterol by monascin, ankaflavin and monacolin K with the sameconcentration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

To achieve the foregoing objectives and effects, the inventors utilize aMonascus fermented product as an extraction substrate, and extract theMonascus fermented product by a series of specific processes, underconstant tests and regulations, thus achieving a composition forlowering blood lipid and elevating high-density lipoprotein and a methodfor manufacturing the same of the present invention. Hereinafter, thecomposition for lowering blood lipid and elevating high-densitylipoprotein and the method for manufacturing the same according to afirst, a second and a third preferred embodiment of the presentinvention are described in detail to illustrate the ingredients of thecomposition and the method for manufacturing the same of the presentinvention.

Before introducing the preferred embodiments of the present invention,the manufacturing method of a Monascus fermented product should beillustrated first. Referring to FIG. 1, which is a flow chart diagram ofthe method for manufacturing the Monascus fermented product of thepresent invention. The method comprises the steps of: (step 001)providing a substrate, which can be rice or dioscorea, wherein the watercontent of the substrate should be below 15%, and the optimal watercontent is 7%; (step 002) adding water to the substrate to make theratio of the substrate to water be 1:0.5%˜1:1.5%, wherein the optimalratio is 1:0.75%; (step 003) soaking the substrate in water, wherein thesoaking time is 0˜60 min, and the optimal time is 30 min; (step 004)sterilizing the substrate, wherein the sterilization condition is 121°C. for 10˜60 min; (step 005) cooling the substrate down to form aculture medium; (step 006) inoculating a specific Monascus spp. to theculture medium, wherein the specific Monascus spp. can be Monascuspurpureus; (step 007) culturing the culture medium inoculated with thespecific Monascus spp., wherein the culture medium is cultured under atemperature of 25˜37° C., a humidity of 50˜80%, and for 8˜20 days, andthe optimal culture condition is the temperature of 30, the humidity of60%, and for 10 days; (step 008) drying the Munascus fermented productcultured in the previous step to make the Munascus fermented producthave a water content below 15%, wherein the optimal water content is 6%.By the above processes, the Monascus fermented product is manufactured.

In the method for manufacturing the Monascus fermented product of thepresent invention, the strains of Monascus purpureus can be selectedfrom Monascus purpureus NTU 568 or Monascus purpureus PAN 790.

Monascus purpureus NTU 568 is deposited in Agricultural Research CultureCollection (NRRL) in Nov. 13, 2009, and the given accession number isNRRL 50338. The characteristics of Monascus purpureus NTU 568 includesrapid growth, strong ability of starch hydrolysis, and strong ability ofmetabolites production. The basic culture manner of Monascus purpureusNTU 568 is to be cultured with a medium containing 2% of rice powder,the best culture temperature is 30° C., the best culture time is 48hours, the best culture pressure is 1 atm, and the growth of Monascuspurpureus NTU 568 is oxygen dependent.

Monascus purpureus PAN 790 is deposited in Agricultural Research CultureCollection (NRRL) in Nov. 13, 2009, and the given accession number isNRRL 50337. The characteristics of Monascus purpureus PAN 790 includesslow growth, being able to produce large amount of monacolin K, andbeing mutated in and cloned from red mold rice. The culture medium forMonascus purpureus PAN 790 includes 2% of rice powder; the culturetemperature is 30° C.; the culture time is 48 hours; the culturepressure is 1 atm; the sterilization manner for Monascus purpureus PAN790 is 121° C. for 20 min; the pH value before sterilization is 5; thegrowth of Monascus purpureus PAN 790 is oxygen dependent; and thestorage temperature for Monascus purpureus PAN 790 is 4° C.

Subsequently, the first preferred embodiment of the present invention isdescribed in detail. The composition for lowering blood lipid andelevating high-density lipoprotein according to the first preferredembodiment of the present invention contains monascin, which is aMonascus yellow pigment, and extracted from the above-mentioned Monascusfermented product. Wherein an amount of the monascin taken by an adultis more than 2.4 mg per day, so that the concentration of blood lipidcan be lowered and the concentration of the high-density lipoprotein inblood can be elevated effectively.

Referring to FIG. 2, which is a flow chart diagram of a method formanufacturing the composition for lowering blood lipid and elevatinghigh-density lipoprotein according to the first preferred embodiment ofthe present invention. The method includes the steps of: (step 101)providing the Monascus fermented product; (step 102) treating theMonascus fermented product with acetone for three times, wherein theratio of the Monascus fermented product to the acetone is 1:10˜1:50;(step 103) elevating the concentration of the product obtained from theprevious step by a process of decompress concentration in a specifictemperature range, wherein the specific temperature range is 40˜60;(step 104) separating a pigment fraction from the product obtained fromthe previous step by a silica gel column chromatography; (step 105)separating a yellow pigment fraction from the pigment fraction by aSephadex LH-20 column chromatography; (step 106) separating a fractioncontaining monascin and ankaflavin from the yellow pigment fraction bythe silica gel column chromatography; and (step 107) separating monascinfrom the fraction containing monascin and ankaflavin obtained from theprevious step by a preparative high performance liquid chromatography(pre-HPLC).

Referring to FIG. 3, which is a flow chart diagram of detailed steps ofstep 104 according to the first preferred embodiment of the presentinvention. The above step 104 can be subdivided into the following stepsof: (step 104 a) adding the product obtained from step 103 to a silicagel column; (step 104 b) adding a plurality of washing solutions to thesilica gel column sequentially, wherein the plurality of washingsolutions are Hexane, Hexane:Ethanol=9:1, Hexane:Ethanol=8:2,Hexane:Ethanol=7:3, Hexane:Ethanol=1:1, and Ethanol sequentially; (step104 c) collecting 12˜15 fractions flowing out from the silica gelcolumn, wherein the time period of each fraction flowing out from thesilica gel column is 5˜10 min; (step 104 d) analyzing the fractionscollected in the previous step by a high performance liquidchromatography (HPLC) in combination with a photodiode-array (PDA), andcollecting fractions containing monascin and ankaflavin; and (step 104e) mixing the fractions collected in the previous step to form thepigment fraction.

Referring to FIG. 4, which is a flow chart diagram of detailed steps ofstep 105 according to the first preferred embodiment of the presentinvention. The above step 105 can be subdivided into the following stepsof: (step 105 a) adding the pigment fraction obtained from step 104 to aSephadex LH-20 column; (step 105 b) adding a washing solution to theSephadex LH-20 column, wherein the washing solution isMethanol:Acetonitrile=9:1; (step 105 c) collecting 3˜5 fractions flowingout from the Sephadex LH-20 column, wherein the time period of eachfraction flowing out from the silica gel column is 5˜10 min; (step 105d) analyzing the fractions collected in the previous step by a HPLC-PDA,and collecting fractions containing monascin and ankaflavin; and (step105 e) mixing the fractions collected in the previous step to form theyellow pigment fraction.

Referring to FIG. 5, which is a flow chart diagram of detailed steps ofstep 106 according to the first preferred embodiment of the presentinvention. The above step 106 can be subdivided into the following stepsof: (step 106 a) adding the yellow pigment fraction obtained from step105 to a silica gel column; (step 106 b) adding a plurality of washingsolutions to the silica gel column sequentially, wherein the pluralityof washing solutions are Dichloromethane:Ethanol=95:5,Dichloromethane:Ethanol=9:1, and Dichloromethane:Ethanol=4:1sequentially; (step 106 c) collecting 3˜5 fractions flowing out from thesilica gel column, wherein the time period of each fraction flowing outfrom the silica gel column is 5˜10 min; (step 106 d) analyzing thefractions collected in the previous step by a HPLC-PDA, and collectingfractions containing monascin and ankaflavin; and (step 106 e) mixingthe fractions collected in the previous step to form the fractioncontaining monascin and ankaflavin.

Referring to FIG. 6, which is a flow chart diagram of detailed steps ofstep 107 according to the first preferred embodiment of the presentinvention. The above step 107 can be subdivided into the following stepsof: (step 107 a) washing and separating the fraction containing monascinand ankaflavin obtained from step 106 by a C₁₈ column in combinationwith a HPLC and a washing solution, wherein the washing solution isMethanol:water=85:15; (step 107 b) collecting 2 fractions flowing outfrom the C₁₈ column, wherein the time period of each fraction flowingout from the silica gel column is 5˜10 min; and (step 107 c) analyzingthe fractions collected in the previous step by a HPLC-PDA, andcollecting fractions containing monascin.

In the above steps 104 d, 105 d and 106 d, the column applied in theHPLC process is C18 column (25 cm*4.6 mm, i.d.=5 μm), the mobile phaseis Acetonitrile:water:trifluoroacetate=62:38:0.05, the speed of flow is1 mL/min, and the UV absorption amount under 231 nm is detected with amultiwavelength detector (because of the curves of monascin andankaflavin have peaks under 231 nm). Furthermore, standards of monascinand ankaflavin should be applied as comparison groups for comparing thepeaks with the ones of analytes, so as to determine whether the analytesare monascin and ankaflavin or not.

Additionally, in the above-mentioned step 107 c, the analysis conditionsof HPLC process are almost the same as step 104 d, step 105 d and step106 d. However, the special aspect of step 107 c is that monascin andankaflavin are separated from each other by the property of polaritydifference. Owing to the C₁₈ column can wash out the more polaringredients at earlier time point, and monascin is more polar relativeto ankaflavin, monascin can be washed out from C₁₈ column earlier thanankaflavin. By this property, monascin can be collected in the fractionswashed out at earlier time point, and monascin in the fractions can beidentified by comparing the washing time and the absorption wavelengthwith the monascin standard.

Subsequently, the second preferred embodiment of the present inventionis described in detail. The composition for lowering blood lipid andelevating high-density lipoprotein according to the second preferredembodiment of the present invention contains ankaflavin, which is aMonascus yellow pigment, and extracted from the above-mentioned Monascusfermented product. Wherein an amount of the ankaflavin taken by an adultis more than 0.6 mg per day, so that the concentration of blood lipidcan be lowered and the concentration of the high-density lipoprotein inblood can be elevated effectively.

Referring to FIG. 7, which is a flow chart diagram of a method formanufacturing the composition for lowering blood lipid and elevatinghigh-density lipoprotein according to the second preferred embodiment ofthe present invention. Steps 201˜206 of the second preferred embodimentare almost the same as the steps 101˜106, and thus the steps 201˜206will not be described again here. The difference between the secondpreferred embodiment and the first preferred embodiment is that step 207of the second preferred embodiment separates ankaflavin by the pre-HPLCprocess.

Referring to FIG. 8, which a flow chart diagram of detailed steps ofstep 207 according to the second preferred embodiment of the presentinvention. The above step 207 can be subdivided into the following stepsof: (step 207 a) washing and separating the fraction containing monascinand ankaflavin obtained from step 206 by a C₁₈ column in combinationwith a HPLC and a washing solution, wherein the washing solution in step(7.1) is Methanol:water=85:15; (Step 207 b) collecting 2 fractionsflowing out from the C₁₈ column, wherein the time period of eachfraction flowing out from the silica gel column is 5˜10 min; and (step207 c) analyzing the fractions collected in the previous step by aHPLC-PDA, and collecting fractions containing ankaflavin.

In the above-mentioned step 207 c, monascin and ankaflavin are separatedfrom each other by the property of polarity difference. Owing to the C₁₈column can wash out the more polar ingredients at earlier time point,and monascin is more polar relative to ankaflavin, monascin can bewashed out from C₁₈ column earlier than ankaflavin. By this property,ankaflavin can be collected in the fractions washed out at later timepoint, and ankaflavin in the fractions can be identified by comparingthe washing time and the absorption wavelength with the ankaflavinstandard.

Subsequently, the third preferred embodiment of the present invention isdescribed in detail. The composition for lowering blood lipid andelevating high-density lipoprotein according to the third preferredembodiment of the present invention contains monascin and ankaflavin,which are both Monascus yellow pigments, and extracted from theabove-mentioned Monascus fermented product. Wherein the content ratio ofthe monascin to the ankaflavin is in a range from 1:1 to 10:1, and theoptimum content ratio of the monascin to the ankaflavin is 3.56:1.Furthermore, an amount of the monascin taken by an adult is more than2.4 mg per day, and an amount of the ankaflavin taken by an adult ismore than 0.6 mg per day, so that the concentration of blood lipid canbe lowered and the concentration of the high-density lipoprotein inblood can be elevated effectively.

Referring to FIG. 9, which is a flow chart diagram of a method formanufacturing the composition for lowering blood lipid and elevatinghigh-density lipoprotein according to the third preferred embodiment ofthe present invention. Steps 301˜306 of the third preferred embodimentare almost the same as the steps 101˜106 and 201˜206, and thus the steps301˜306 will not be described again here. The difference between thethird preferred embodiment and the first preferred embodiment (or secondpreferred embodiment) is that step 307 of the third preferred embodimentseparates monascin and ankaflavin by the pre-HPLC process. Eventually,according to step 308, mixing monascin and ankaflavin together accordingto a specific ratio, wherein the specific ratio is in a range from 1:1to 10:1, and the optimum value of the specific ratio is 3.56:1.

Referring to FIG. 10, which is a flow chart diagram of detailed steps ofstep 307 according to the third preferred embodiment of the presentinvention. The above step 307 can be subdivided into the following stepsof: (step 307 a) washing and separating the fraction containing monascinand ankaflavin obtained from step 306 by a C₁₈ column in combinationwith a HPLC and a washing solution, wherein the washing solution isMethanol:water=85:15; (step 307 b) collecting 2 fractions flowing outfrom the C₁₈ column, wherein the time period of each fraction flowingout from the C₁₈ column is about 5˜10 min; and (step 307 c) analyzingthe fractions collected in the previous step by a HPLC-PDA, andcollecting fractions containing monascin and ankaflavin respectively.

In the above-mentioned step 307 c, monascin and ankaflavin are separatedfrom each other by the property of polarity difference. Owing to the C₁₈column can wash out the more polar ingredients at earlier time point,and monascin is more polar relative to ankaflavin, monascin can bewashed out from C₁₈ column earlier than ankaflavin. By this property,monascin can be collected in the fractions washed out at earlier timepoint, and ankaflavin can be collected in the fractions washed out atlater time point. Furthermore, monascin and ankaflavin in the fractionscan be identified by comparing the washing time and the absorptionwavelength with the monascin and ankaflavin standards.

In order to prove that the compositions of the first, the second and thethird preferred embodiments indeed have the effect of lowering bloodlipid and elevating high-density lipoprotein, the compositions areanalyzed and compared for their functions by several experiments asfollowings. Hereinafter, the composition of the first preferredembodiment is called as monascin (MS for abbreviation), and the amountof the monascin taken by an experimental animal is equivalent to 9.82 mgtaken by an adult human per day; the composition of the second preferredembodiment is called as ankaflavin (AF for abbreviation), and the amountof the ankaflavin taken by an experimental animal is equivalent to 1.43mg taken by an adult human per day; the composition of the thirdpreferred embodiment is called as monascin+ankaflavin (MS+AF forabbreviation), and the amount of the monascin and ankaflavin taken by anexperimental animal are equivalent to 9.82 mg and 1.43 mg taken by anadult human per day respectively. Additionally, a composition containingmonacolin K is utilized as a comparison group, which is called as MK forabbreviation.

Firstly, the experiment utilizes hamsters feed with high cholesteroldiet as experimental animals, which are then feed with MS, AF, MS+AF,and MK for 8 weeks, and then each of cholesterol parameters in blood ofthe hamsters are analyzed. The cholesterol parameters include totalcholesterol (TC), triglyceride (TG), high density lipoproteincholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), ratioof LDL-C to HDL-C, and malondialdehyde (MDA). The amount of the MK takenby the experimental animals is equivalent to 2.89 mg taken by an adulthuman per day. Additionally, a normal diet group is referred to as NOR,and a high cholesterol diet group is referred to as HC.

Referring to the following table 1 and table 2, wherein the table 1illustrates the results of the above-mentioned animal experiments, andthe table 2 illustrates the reducing rate or the increasing rate of MS,AF, MS+AF, and MK relative to HC. In the results of the reducing rate ofTC, the groups of MS and AF both can lower the amount of TC in serumsignificantly, and the effect of MS and AF are better than that of MK;the group of MS+AF has the effect of lowering the amount of TC betterthan the other groups. In the results of the reducing rate of TG, groupsof MS, AF and MK+AF can lower the amount of TG in serum significantly,and the effect of MS, AF and MS+AF are better than that of MK. In theresults of the increasing rate of HDL-C, the groups of MS and AF bothcan increase the amount of TC in serum significantly, and the effect ofMS and AF are better than that of MK; the group of MS+AF has the effectof increasing the amount of HDL-C better than the other groups. In theresults of the reducing rate of LDL-C, although the group of MS has theeffect almost the same as MK, the groups of AF and MS+AF have the effectof lowering the amount of LDL-C better than the MK group. In the resultsof the reducing rate of LDL/HDL, the effect of MS group is better thanMK group, and the effects of AF and MS+AF are better than the othergroups, thus monascin, ankaflavin and monascin+ankaflavin is morecontributive to ameliorate the occurrence of cardiovascular diseases andatherosclerosis than monacolin K. In the results of the reducing rate ofMDA, the groups of MS, AF and MS+AF can lower the amount of MDA in serumsignificantly, and the effects of the groups of MS, AF and MS+AF are allbetter than the MK group.

TABLE 1 TC TG HDL-C LDL-C MDA content content content content contentGroup (mg/dL) (mg/dL) (mg/dL) (mg/dL) LDL/HDL (mg/dL) NOR 111.8 ± 168.8± 66.6 ± 19.9 ± 0.278 ± 8.93 ± 10.7 35.6 5.97 2.59 0.038 1.79 HC 236.5 ±226.3 ± 98.1 ± 68.3 ± 0.569 ± 11.80 ± 18.9 76.5 8.89 9.75 0.093 1.85 MK190.3 ± 131.1 ± 96.9 ± 45.0 ± 0.489 ± 10.37 ± 25.1 36.7 8.84 6.97 0.0501.73 MS 165.8 ± 82.8 ± 114.2 ± 45.1 ± 0.416 ± 8.62 ± 10.6 9.0 9.36 3.360.022 1.45 AF 168.9 ± 94.5 ± 118.6 ± 39.4 ± 0.355 ± 9.07 ± 11.5 18.08.09 5.80 0.049 1.45 MS + AF 160.3 ± 85.6 ± 125.7 ± 38.5 ± 0.351 ± 8.45± 7.6 10.60 7.30 3.10 0.051 1.23

TABLE 2 TC TG HDL-C LDL-C LDL/HDL MDA reducing reducing increasingreducing reducing reducing group rate (%) rate (%) rate (%) rate (%)rate (%) rate (%) MK 19.6 42.1 −1.2 34.1 14.1 16.5 MS 29.9 63.4 14.133.9 26.9 40.1 AF 28.6 58.2 17.3 42.3 37.6 59.6 MS + AF 29.2 63.8 22.043.6 38.3 58.9

Another important indicator for evaluating atherosclerosis is theanalysis of lipid plaque staining in arteries, and the results of lipidplaque staining can indicate that whether lipid plaque deposits onarterial vascular walls. Large amounts of lipid with over-oxidation cancause bulk deposition of foam cells, and then the vascular walls loseelasticity and large amounts of lipid plaque are deposited, so as tolead to atherosclerosis. Referring to FIG. 11, which is a diagram ofquantitative results of the lipid plaque staining experiment. Theresults demonstrate that high cholesterol diet (HC group) for eightweeks may cause the experimental animals to deposit large amounts oflipid plaque on arterial vascular walls in the thoracic cavity, but thedeposition of lipid plaque in the NOR group is less than HC group.Although the MK group (the amount of the MK taken is 2.89 mg taken by anadult human per day) can lower the amount of lipid plaque, the effect ofthe MK group is not as good as that of the MS (the amount of the MStaken is 9.82 mg taken by an adult human per day), AF (the amount of theAF taken is 1.43 mg taken by an adult human per day) and MS+AF groups(the content ratio of the monascin to the ankaflavin is 3.56:1); theeffect of the AF group is better than that of the MS group, and theeffect of the MS+AF group is better than that of the AF group.

In order to compare the inhibitory effect of cholesterol biosynthesis bymonascin, ankaflavin and monacolin K, the present invention applies acell experiment and treats cells with monascin, ankaflavin and monacolinK with the same concentration (100 ppb) for researching the influence ofthese ingredients on the cholesterol biosynthesis. Referring to FIG. 12,which is a comparative diagram for the effect of the inhibition ofcholesterol by monascin, ankaflavin and monacolin K with the sameconcentration. The results demonstrate that ankaflavin has the besteffect of inhibiting the biosynthesis of cholesterol, followed bymonascin, and monacolin K has the weakest effect. These results areconsistent with the above animal experiments, that is ankaflavin andmonascin have better effect on blood lipid lowering than monacolin K.

It can be known from the above experiments that monascin, ankaflavin andmonascin+ankaflavin can reduce TC, TG, LDL-C, MDA, and lipid plaquesignificantly, and can elevate the content of HDL-C in serum;additionally, the effect of monascin+ankaflavin is more prominent thanthe effect of monascin or ankaflavin. Thus, the compositions accordingto the first, second and third preferred embodiments not only can reducethe content of cholesterol in serum, but also can elevate theconcentration of HDL-C in serum, so as to have extreme contribution tolower the incidence of cardiovascular diseases and atherosclerosis.

By the detailed description of the overall structure and technicalcontent of the present invention, the following advantages of thepresent invention can be derived:

-   1. Each of the compositions provided by the present invention all    have the effect of lowering blood lipid and elevating high-density    lipoprotein, thus these compositions can be utilized to prevent and    treat cardiovascular diseases and atherosclerosis.-   2. Through the manufacturing method provided by the present    invention, each of the compositions with high purity of the present    invention can be extracted, and can be applied to lower the    incidence of cardiovascular diseases and atherosclerosis.-   3. Each of the compositions of the present invention are extracted    from Monascus fermented product, not synthesized artificially, thus    the compositions can be regarded as natural food supplements, which    not only can reduce the incidence of cardiovascular diseases and    atherosclerosis, but also can avoid the production of side effects.

It should be understood that the embodiments of the present inventiondescribed herein are merely illustrative of the technical concepts andfeatures of the present invention and are not meant to limit the scopeof the invention. Those skilled in the art, after reading the presentdisclosure, will know how to practice the invention. Various variationsor modifications can be made without departing from the spirit of theinvention. All such equivalent variations and modifications are intendedto be included within the scope of the invention.

As a result of continued thinking about the invention and modifications,the inventors finally work out the designs of the present invention thathas many advantages as described above. The present invention meets therequirements for an invention patent, and the application for a patentis duly filed accordingly. It is expected that the invention could beexamined at an early date and granted so as to protect the rights of theinventors.

What is claimed is:
 1. A method for manufacturing a composition forlowering blood lipid and elevating high-density lipoprotein, wherein themethod comprises the steps of: (1) providing a Monascus fermentedproduct; (2) treating the Monascus fermented product with acetone forthree times, wherein the ratio of the Monascus fermented product to theacetone is 1:10˜1:50; (3) elevating the concentration of the productobtained from the step (2) by a process of decompress concentration in aspecific temperature range, wherein the specific temperature range instep (3) is 40° C.˜60° C.; (4.1) adding the product obtained from thestep (3) to one silica gel column; (4.2) adding a plurality of washingsolutions to the silica gel column sequentially, wherein the pluralityof washing solutions are sequentially hexane, hexane ethanol mixturewith the volume ratio of hexane to ethanol being nine to 1 (9:1), hexaneethanol mixture with the volume ratio of hexane to ethanol being eightto two (8:2), hexane ethanol mixture with the volume ratio of hexane toethanol being seven to three (7:3), hexane ethanol mixture with thevolume ratio of hexane to ethanol being five to five (5:5), and ethanol;(4.3) collecting 12˜15 fractions flowing out from the silica gel column;(4.4) analyzing the fractions collected in the previous step by a highperformance liquid chromatography (HPLC) in combination with aphotodiode-array (PDA), and then collecting fractions containingmonascin and ankaflavin; (4.5) mixing the fractions collected in thestep (4.4) to form a pigment fraction; (5.1) adding the pigment fractionobtained from the step (4.5) to a Sephadex LH-20 column; (5.2) adding awashing solution to the Sephadex LH-20 column, wherein the washingsolution is a methanol acetonitrile mixture with the volume ratio ofmethanol to acetonitrile being nine to one (9:1); (5.3) collecting 3˜5fractions flowing out from the Sephadex LH-20 column; (5.4) analyzingthe fractions collected in the step (5.3) by a HPLC in combination witha PDA, and then collecting fractions containing monascin and ankaflavin;(5.5) mixing the fractions collected in the step (5.4) to form a yellowpigment fraction; (6.1) adding the yellow pigment fraction obtained fromthe step (5.5) to one silica gel column; (6.2) adding a plurality ofwashing solutions to the silica gel column sequentially wherein theplurality of washing solutions are sequentially dichloromethane,dichloromethane ethanol mixture with the volume ratio of dichloromethaneto ethanol being ninety five to five (95:5), dichloromethane ethanolmixture with the volume ratio of dichloromethane to ethanol being nineto one (9:1), and dichloromethane ethanol mixture with the volume ratioof dichloromethane to ethanol being four to one (4:1); (6.3) collecting3˜5 fractions flowing out from the silica gel column; (6.4) analyzingthe fractions collected in the previous step by a HPLC in combinationwith a PDA, and then collecting fractions containing monascin andankaflavin; (6.5) mixing the fractions collected in the step (6.5) toform a specific fraction containing high-purity monascin and high-purityankaflavin; (7.1) washing and separating the specific fractioncontaining monascin and ankaflavin obtained from the step (6.5) througha C₁₈ column in combination with a high performance liquidchromatography (HPLC); (7.2) collecting two fractions flowing out fromthe C₁₈ column, wherein the washing solution is a methanol water mixturewith the volume ratio of methanol to water is eighty five to fifteen(85:15); and (7.3) analyzing the two fractions collected in the step(7.2) by using a HPLC in combination with a PDA, so as to determine andcollect the fraction of monascin and the fraction of ankaflavin; and (8)mixing the monascin and the ankaflavin, wherein the ratio of monascin toanfaflavin ranges from 1:1 to 10:1.
 2. The method for manufacturing thecomposition for lowering blood lipid and elevating high-densitylipoprotein according to claim 1, wherein the optimum value of the ratioof monascin to ankaflavin in the step (8) is 3.56:1.
 3. The method formanufacturing the composition for lowering blood lipid and elevatinghigh-density lipoprotein according to claim 1, wherein the compositioncomprises monascin and ankaflavin, and both the monascin and theankaflavin are a Monascus yellow pigment extracted from thr Monascusfermented product, wherein the content ratio of the monascin to theankaflavin is ranged from 1:1 to 10:1.
 4. The method for manufacturingthe composition for lowering blood lipid and elevating high-densitylipoprotein according to claim 3, wherein the optimum content ratio ofthe monascin to the ankaflavin is 3.56:1.
 5. The method formanufacturing the composition for lowering blood lipid and elevatinghigh-density lipoprotein according to claim 3, wherein a daily dosecomposition for an adult comprises an amount of the monascin more than2.4 mg, and an amount of the ankaflavin more than 0.6 mg.